The requirements in cis for DNA to serve as an origin of DNA synthesis, that is, an initiator, in metazoan cells are not well defined. Experimental findings have indicated there may be no requirements under some conditions (Harland and Laskey, 1980) while other findings have mapped an origin to specific nucleotides (Abdurashidova, 2000), a result consistent with a specific DNA sequence contributing to an origin of synthesis. The Epstein-Barr Viral (EBV) origin of plasmid DNA synthesis, oriP, efficiently supports licensed DNA synthesis in a variety of higher eukaryotic cells. This origin uses only one viral protein, EBNA-1, while all other factors are provided by the cell. It thus serves as one model for characterizing cis-acting requirements for a licensed, metazoan origin of DNA synthesis.
The origin of DNA synthesis within oriP is the Dyad Symmetry (DS) element, which has been genetically dissected to identify several of its key features. DS is composed of two pairs of binding sites for its only required viral protein EBNA-1, three 9 bp elements that resemble the telomeric repeats of the ends of human chromosomes and are half-binding sites for TTAGGG-repeat Binding Factor 2 (TRF2), a dyad element from which the name was derived, and a region upstream of DS that was found to be helically unstable (Baer et al., 1984; Bashaw and Yates, 2001; Deng et al., 2002; Niller et al., 1985; Polonskaya et al., 2004; Rawlins et al., 1985; Yates et al., 2000). Of these elements, a pair of appropriately spaced EBNA-1-binding sites was found to be the minimal cis-acting element required for the replicative function of DS (Bashaw and Yates, 2001).
The DS element recruits multiple licensing factors for DNA replication (e.g. ORC1-6, MCM2-7, Cdt1, Geminin), which function indistinguishably from the roles assigned to them with human chromosomes (Chaudhuri et al., 2001; Dhar et al., 2001; Julien et al., 2004; Ritzi et al., 2003; Shepers et al., 2001). The DS element recruits these proteins either directly by serving as a substrate for sequence-specific binding, as in the case of TRF2, or indirectly through proteins that bind it site-specifically, and also bind additional proteins. TRF2, for example, can bind ORC1 (Atanasiu et al., 2006). EBNA-1 has an N-terminal domain that is required for the synthesis of oriP and mimics the human HMGA1a protein in both its overall amino acid composition and it's AT-hook sequence motifs (Hung et al., 2001; Sears et al., 2004). This molecular mimicry has been demonstrated by the finding that HMGA1a fused to the DNA-binding and dimerization domain of EBNA-1 restores the ability to support DNA synthesis to this derivative of EBNA-1 (Hung et al., 2001; Sears et al., 2004; Altman et al., 2006). These experiments showed that the N-terminal domain of EBNA-1 and HMGA1a both confer origin function to DS when bound by it site-specifically.
EBV has a second plasmid origin of DNA synthesis, Rep*, which has been experimentally compared to DS (Wang et al., 2006). Rep* contains two EBNA-1-binding sites with 21 bp center-to-center spacing, which, as with DS, is critical for pre-replication complex (preRC) recruitment and replicative function (Wang et al., 2006). However, Rep* does not contain a dyad, TRF2-binding sites, nor a helically unstable region upstream of it, indicating that these elements are not essential for the initiation of DNA synthesis and at most may play auxiliary roles in this process (Wang et al., 2006).